Graft-versus-host disease (GvHD) is a potentially life threatening complication following allogeneic hematopoietic stem cell transplantation (HSCT). JAK2 inhibition induces allotolerance, while preserving Treg development. Preliminary data supports that JAK2 inhibition combined with IL-2 offers a synergistic effect on suppressing alloreactivity. Moreover, blockade of downstream STAT3 abrogates alloresponses as a single agent. These strategies preserve Treg-dependent IL-2/STAT5 signaling, increasing the ratio of STAT5 to STAT3 phosphorylation. The proposed experiments will investigate the influence of JAK2 inhibition paired with IL-2, or STAT3 blockade alone; on the control of allosensitization. This work is highly relevant to the mission of the NHLBI, as it will offer new insights on eliminating the profound morbidity and mortality of GvHD. The proposed experiments will evaluate the following specific aims: Aim 1) Examine JAK2 inhibition paired with IL-2, or STAT3 blockade alone, as a platform to control alloreactivity and optimize Treg expansion in vitro. Aim 2) Investigate the feasibility, biologic influence, and therapeutic effect of skewing the STAT5/STAT3 phosphorylation ratio in a mouse model of GVHD and GVL. Aim 3) Determine if the pSTAT5/pSTAT3 ratio or ERK1/2 phosphorylation in CD4+ or CD8+ T cells on day +21 associate with the development of acute GvHD before day +100. Dendritic cell (DC)-allostimulated T cells will be used to investigate the influence of pSTAT5/pSTAT3 polarization via JAK2 inhibition combined with IL-2, or STAT3 inhibition alone, as a means to suppress alloreactivity and promote Treg expansion. Mechanistic studies will evaluate secondary effects on Treg suppression, STAT signaling, and skewing of T helper subsets and related functions. The resultant data will be confirmed by molecularly targeting STAT3 with siRNA. This concept will be tested in vivo with a GVHD prophylaxis regimen of a JAK2 and IL-2, or STAT3 inhibitor alone, in an MHC- mismatched mouse model (C57BL/6 => Balb/c). Mice will be assessed for GVHD clinical scores, survival, and alterations in Treg/Th1/Th17 subsets. The concept of STAT5/STAT3 and ERK1/2 phosphorylation as biomarkers of impending acute GvHD will be investigated in a 110 patient sample study. JAK2-mediated STAT3 and parallel ERK1/2 phosphorylation will be measured in patients' T cells on day +21 after allogeneic HSCT. The ratio of STAT5/STAT3 phosphorylation will be studied for any protective influence on GvHD. Logical extensions of this work will focus on translating the concept of JAK2 paired with IL-2, or STAT3 blockade alone, in novel clinical trials for GVHD prevention and therapy. The informative data provided by aim 3, if positive, will be validated in a larger patient population to determine f the STAT3, STAT5, and ERK1/2 phosphorylation predict the risk of acute GvHD.